A conserved ZFX/WNT3 axis modulates the growth and imatinib response of chronic myeloid leukemia stem/progenitor cells

Background Zinc finger protein X-linked (ZFX) has been shown to promote the growth of tumor cells, including leukemic cells. However, the role of ZFX in the growth and drug response of chronic myeloid leukemia (CML) stem/progenitor cells remains unclear. Methods Real-time quantitative PCR (RT–qPCR) and immunofluorescence were used to analyze the expression of ZFX and WNT3 in CML CD34+ cells compared with normal control cells. Short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) technologies were used to study the role of ZFX in growth and drug response of CML cells. Microarray data were generated to compare ZFX-silenced CML CD34+ cells with their controls. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to study the molecular mechanisms of ZFX to regulate WNT3 expression. RT–qPCR and western blotting were used to study the effect of ZFX on β-catenin signaling. Results We showed that ZFX expression was significantly higher in CML CD34+ cells than in control cells. Overexpression and gene silencing experiments indicated that ZFX promoted the in vitro growth of CML cells, conferred imatinib mesylate (IM) resistance to these cells, and enhanced BCR/ABL-induced malignant transformation. Microarray data and subsequent validation revealed that WNT3 transcription was conservatively regulated by ZFX. WNT3 was highly expressed in CML CD34+ cells, and WNT3 regulated the growth and IM response of these cells similarly to ZFX. Moreover, WNT3 overexpression partially rescued ZFX silencing-induced growth inhibition and IM hypersensitivity. ZFX silencing decreased WNT3/β-catenin signaling, including c-MYC and CCND1 expression. Conclusion The present study identified a novel ZFX/WNT3 axis that modulates the growth and IM response of CML stem/progenitor cells. Supplementary Information The online version contains supplementary material available at 10.1186/s11658-023-00496-z.


Table S2
The shRNA sequences used in this study.

Table S3
The gene-specific primers used in this study.

For WNT3 overexpression
Human WNT3 F TCTAGAATGGAGCCCCACCTGCTCGGGCT

R CATATGCTTGCAGGTGTGCACGTCGTAGATG
The underlined sequences represent restriction endonuclease sites.

Table S4
Antibodies used in this study.

Table S5
Guide RNA used in this study.

Table S6
Differentially expressed transcripts between ZFX-silenced CML CD34 + cells and their controls.

Fig. S1
Fig. S1 Zinc finger protein X-linked is upregulated in chronic myeloid leukemia cells and decreases upon imatinib methylate treatment.A The expression of zinc finger protein X-linked (ZFX) in unfractioned BMCs and CD34 + cells from healthy donors and CML patients was assessed with RT-qPCR.B The expression of Zfx in BaF3 cells upon BCR/ABL transduction was measured by RT-qPCR.C The expression of ZFX in NBM CD34 + cells upon BCR/ABL overexpression was assessed by RT-qPCR.D The expression of Zfx upon BCR/ABL transduction in BaF3 cells was analyzed by Western blotting.E K562 cells were treated with various concentrations of imatinib methylate (IM), and ZFX expression was detected by Western Blotting.F The expression of ZFX in CD34 + cells from CML patient in chronic phase with or without IM treatment was analyzed by cofocal microscopy.The representative photos are shown.The scale bar is 20 μm.Data are presented as the mean ± SEM, and Student's t test was used to estimate the P values (*P < 0.05 and **P < 0.01)..

Fig. S2 A
Fig. S2 Zfx overexpression promotes leukemia generation induced by BaF3-BCR/ABL cells.A The weight of the control (Ctrl) and Zfx-overexpressing groups of mice was monitored after tail vein injection.B The diseased mice from the control and Zfxoverexpressing groups were dissected, and leukemic cells were collected and subjected to RT-qPCR analysis for Zfx expression.C The cells from the bone marrow (BM), peripheral blood (PB), and spleen of both the control and Zfx-overexpressing group were analyzed by flow cytomety to detect the infiltration of BaF3-BCR/ABL cells.The representative flow cytometry profiles are displayed (left panel) and the infiltration of BaF3-BCR/ABL cells were statistically analyzed (right panel).D The typical photos of the liver and spleen of the diseased mice from both groups are displayed (left panel).The coefficients of the liver and spleen of both groups were calculated and compared (right panel).Data are presented as the mean ± SEM, and Student's t test was used to estimate the P values (*P < 0.05).

Fig. S4
Fig. S4 ZFX silencing promtes Imatinib mesylate induced cell death of K562 cells.The control (scramble) and ZFX-silenced (shZFX) K562 cells were treated with or without imatinib mesylate (IM) and analyzed by Annexin V/7-AAD staining.A The representative flow cytomety profiles are displayed.B The percentage of apoptotic cells (Annexin V + ) were statistically summarized.Data are presented as the mean ± SEM, and Student's t test was used to estimate the P values (**P < 0.01).

Fig. S5
Fig. S5 Zfx silencing decreases the infiltration of BaF3-BCR/ABL cells.Zfx-difcient and control BaF3-BCR/ABL cells were injected intravenously into the irradiated mice.When the mice were diseased and near death, they were sacrificed and dissected to analyze BaF3-BCR/ABL cell infiltration in bone marrow (BM), peripheral blood (PB), spleen, and liver by flow cytomety.A The representative flow cytometry profiles to analyze the bone marrow and spleen are displayed.B The infiltration of Baf3-BCR/ABL cells were statistically analyzed.Data are presented as the mean ± SEM, and Student's t test was used to estimate the P values.

Fig. S6
Fig. S6 The expression of DIS3L, SMO, and HOXB5 in ZFX-silenced and control CML cells.A, B The expression of DIS3L, SMO, and HOXB5 in ZFX-silenced and control (scramble) CML CD34 + cells (n=3) and K562 cells were assessed with RT-qPCR (n=3).Data are presented as the mean ± SEM, and Student's t test was used to estimate the P values (*P < 0.05 and ** P < 0.01).

Fig. S7
Fig. S7The KEGG analysis of the differentially expressed transcripts comparing ZFXsilenced with control CML CD34 + cells.

Fig. S8
Fig. S8 Chromatin immunoprecipitation (ChIP) was performed to analyze the interaction between ZFX protein and the WNT3 gene.

Fig. S9
Fig. S9 The alignment analysis of ZFX and WNT3 proteins in mammals.A The alignment analysis of ZFX protiens in Human, Mouse, Rat, Cattle, and dog.B The alignment analysis of WNT3 proteins betwenn Human and Mouse is shown (lower panel) and the similarity between Human WNT3 and Mouse Wnt3 is summarized (upper panel).

Fig. S10
Fig. S10 The expression of WNT3 and ZFX in variously transduced K562 and BaF3-BCR/ABL cells.A, B WNT3 and the empty control (Ctrl) were delivered into the control (Scramble) and ZFX-silenced K562 cells (A) and BaF3-BCR/ABL cells (B), the expression of WNT3 and ZFX was analyzed by Western blotting.

Fig. S11
Fig. S11 Activated β-catenin was significant reduced upon ZFX silencing in both K562 and BaF3-BCR/ABL cells.A,B The expression of activated β-catenin in K562 cells (A) and BaF3-BCR/ABL cells (B) was detected by flow cytometric analysis and reanalyzed by FlowJo software.Data are presented as the mean ± SEM, and Student's t test was used to estimate the P values (*P < 0.05 and **P < 0.01).MFI, mean fluorescence intensity.

Fig. S12
Fig. S12 The expression of c-MYC and CCND1 is regulated by ZFX in various cellular models.A, B The expression of c-MYC and CCND1 was measued in ZFX-silenced (shZFX) and control (scramble) CML CD34 + and K562 cells.C The expression of c-Myc and Ccnd1 was measured in Zfx-silenced and control (Scramble) Baf3-BCR/ABL cells.D The expression of c-Myc and Ccnd1 was assessed in Zfx-overexpressing (Zfx) and control (Ctrl) BaF3-BCR/ABL cells.Data are presented as the mean ± SEM, and Student's t test was used to estimate the P values (*P < 0.05, **P < 0.01, and *** P < 0.001).

Table S7
KEGG analysis of signaling pathways among differentially expression transcripts comparing ZFX-silenced CML CD34 + cells with their controls.